The present invention relates to processes for preparing novel cationic ainphiphiles containing N-hydroxyalkyl group. The invention provides novel compositions containing the said amphiphiles that are useful to facilitate transport of biologically active molecules into cells. The area of medical science that is likely to benefit most from the present invention is gene therapy.
In gene therapy, patients carrying identified defective genes are supplemented with the copies of the corresponding normal genes. However, genes (DNA), the polyanionic macromolecules and the cell surfaces of the biological membranes both being negatively charged, spontaneous entry of normal copies of genes into the target cells of patients is an inefficient process (because of electrostatic repulsion). This is why the past decade has witnessed an unprecedented upsurge of global interest in developing efficient gene delivery reagents for introducing normal genes into the target cells of patients suffering from various genetic (inherited) diseases such as cystic fibrosis, Gaucher""s illness, Fabry""s disease etc. Many gene delivery reagents (also known as transfection vectors) including retrovirus, adenovirus, and cationic amphiphilic compounds (i.e. compounds containing both polar and non-polar functionalities) are being used as the carriers of polyanionic genes in combating hereditary diseases in gene therapy. The amphiphilic nature (presence of both polar and non-polar regions in the molecular structures) of the compounds designed to deliver therapeutically actives molecules, ensures smooth interaction of these carrier molecules with the polar and non-polar regions of plasma membranes, compartments within the cells and the biologically active molecules itself. At physiological pH, the cationic amphiphiles in the form of liposomes or micelles associate favorably with the negatively charged regions of the macromolecular polyanionic DNA enhancing the intracelluar uptake of the resulting complex between the cationic lipids and the negatively charged DNA. Reproducibility, high degree of targetability and low cellular toxicity are increasingly making the cationic amphiphiles the transfection vectors of choice in gene therapy.
An impressive number of cationic lipids with varying structures have been reported for the intracellular delivery of therapeutically active molecules as exemplified by the following references:
Felgner et al., Proc. Natl. Acad. Sci. U.S.A., 84, 7413-7417 (1987), reported the first use of a highly efficient cationic lipid N-[1-(2,3-ioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) as the DNA transfection vector.
U.S. Pat. Nos. 4,897,355 and 4,946,787 (1990) reported the synthesis and use of N-[.omega..(.omega.-1)-dialkloxoxy]- and N-[..omega..(.omega.1)-dialkenyloxy]-alk-1 -yl-N,N,N-tetrasubstituted ammonium amphiphiles and their pharmaceutical formulations as efficient transfection vectors.
Leventis, R and Silvius, J. R. Biochim. Biophys. Acta. 1023, 124-132, (1990) reported the interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles.
U.S. Pat Nos. 5,264,618 (1993) reported the synthesis and use of additional series of highly efficient cationic lipids for intracellular delivery of biologically active molecules.
Felgner et al. J. Biol. Chem. 269,2550-2561 (1994) reported enhanced gene delivery and mechanistic studies with a novel series of cationic lipid formulations.
U.S. Pat. No. 5,283,185 (1994) reported the synthesis and use of 3xcex2[N-(N1,N1-dimethylaminoethane)carbamoyl] cholesterol, termed as xe2x80x9cDC-Cholxe2x80x9d for delivery of a plasmid carrying a gene for chloramphenicol acetyl transferase into cultured mammalian cells.
U.S. Pat. No. 5,283,185 (1994) reported the use of N-[2-[[2,5-bis[(3-aminopropyl)amino]-1-Oxopentyl]aminoethyl]-N,N-dimethyl-2,3-bis-(9-Octadecenyloxy)-1-Propanaminium tetra (trifluoroacetate), one of the most widely used cationic lipids in gene delivery. The pharmaceutical formulation containing this cationic lipid is sold commercially under the trade name xe2x80x9cLipofectaminexe2x80x9d.
Solodin et al. Biochemistry 34, 13537-13544, (1995) reported a novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery.
Wheeler et al. Proc. Natl. Acad. Sci. U.S.A. 93, 11454-11459, (1996) reported a novel cationic lipid that greatly enhances plasmid DNA delivery and expression in mouse lung.
U.S. Pat No. 5,527,928 (1996) reported the synthesis and the use of N,N,N,N-tetramethyl-N,N-bis(hydroxy ethyl)-2,3-di(oleolyoxy)-1,4-butanediammonium iodide i.e. pharmaceutical formulation as transfection vector.
U.S. Pat. No. 5,698,721 (1997) reported the synthesis and use of alkyl O-phosphate esters of diacylphosphate compounds such as phosphatidylcholine or phosphatidylethanolamine for intracellular delivery of macromolecules.
U.S. Pat. Nos. 5,661,018; 5,686,620 and 5,688,958 (1997) disclosed a novel class of cationic phospholipids containing phosphotriester derivatives of phosphoglycerides and sphingolipids efficient in the lipofection of nucleic acids.
U.S. Pat. No. 5,614,503 (1997) reported the synthesis and use of an amphipathic transporter for delivery of nucleic acid into cells, comprising an essentially nontoxic, biodegradable cationic compound having a cationic polyamine head group capable of binding a nucleic acid and a cholesterol lipid tail capable of associating with a cellular membrane.
U.S. Pat. No. 5,705,693 (1998) disclosed the method of preparation and use of new cationic lipids and intermediates in their synthesis that are usefild for trasfecting nucleic acids or peptides into prokaryotic or eukaryotic cells. These lipids comprise one or two substituted arginine, lysine or omithine residues, or derivatives thereof, linked to a lipophilic moiety.
U.S. Pat. No. 5,719,131 (1998) has reported the synthesis of a series of novel cationic amphiphiles that facilitate transport of genes into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, alkylamines or polyalkylamines.
Although the above mentioned cationic lipids have been successfully exploited for the intracellular delivery of genes, the efficiencies for the intracellular uptake procedures are insufficient and need to be improved. The transfection activities of most of the above mentioned cationic lipids are modest and therefore substantial quantities of these cationic lipids must be consumed. The associated cellular toxicities of the lipids and the metabolites thereof are, thus naturally, issues of concern Accordingly, demands for developing new class of cationic amphiphiles with high transfection efficiencies and low cellular toxicities continue in this field of art.
The main objective of the present invention is to provide novel simple and economic processes for the synthesis of cationic amphiphilic compounds containing non-toxic N-hydroyalkyl group.
Another objective of the invention is to provide processes for the synthesis of said novel cationic amphiphilic compounds which are useful for delivery of therapeutically effective amounts of biologically active molecules into cells/tissues of patients.
Yet another objective of the invention is to provide novel processes for the synthesis of cationic amphiphilic compounds such that a hydrophobic group is either directly linked to the positively charged Nitrogen atom or is linked to the said Nitrogen atom via an ester or methylene group.
Still another objective of the invention is to provide novel processes for the synthesis of cationic amphiphilic compounds with at least one hydroxyalkyl group containing 1-3 carbon atoms directly linked to the positively charged Nitrogen atom.
Yet another objective of the invention is to provide novel cationic amphiphilic compounds without any glycerol backbone in their structure.
The present invention relates to novel cationic amphiphiles containing nontoxic N-hydroxyalkyl group and provides processes for the preparation said amphiphilic compounds. The novel cationic amphiphiles containing N-hydroxyalkyl group of this invention are potentially useful to deliver into the cells of patients therapeutically effective amounts of biologically active molecules. The area of medical science that is likely to benefit most from the present invention is gene therapy.
Cationic amphiphiles disclosed in the present invention possess several novel structural features. These features may be compared with cationic amphiphilic structures disclosed in Felgner et al. J. Biol. Chem., 269, 2550-2561 (1994), a representative structure of which is 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (xe2x80x9cDMRIExe2x80x9d) and to those disclosed by Bennett et al. J. Med. Chem., 40, 4069-4078 (1997), a representative structure of which is N,N-[Bis(2-hydroxyethyl)]-N-methyl-N-[2,3-bis(tetradecanoyloxy)propyl] ammonium chloride (xe2x80x9cDMDHPxe2x80x9d).
The following distinctive strut features are common to the cationic amphiphiles disclosed in the present invention: (1) the presence of a hydrophobic group which is either directly linked to the positively charged nitrogen atom or is linked to the positively charged nitrogen via an ester group, (2) the presence of at least one hydroxyalkyl group containing 1-3 carbon atom that is directly linked to the positively charged nitrogen atom and (3) unlike many other glycerol-based cationic amphiphiles, the cationic transfection lipids disclosed in the present invention do not have any glycerol backbone in their molecular architecture. It is believed that these unique structural features contribute significantly to the increased transfection efficiencies of the cationic amphiphiles disclosed herein. The enhanced in vitro transfection efficiencies of N,N,di-[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N-methylammonium bromide (DOHEMAB) and N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB), the two novel transfection lipids of the present invention, is compared to Lipofectamine, the most widely used commercially available highly efficient transfection lipid.
According to the practice of the present invention, xe2x80x9ccationicxe2x80x9d means the positive charge is either on a quaternized nitrogen atom or on a protonated tertiary nitrogen atom. The cationic characters of the amphiphiles may contribute to the enhanced interaction of the amphiphiles with biologically active molecules (such as nucleic acids) and/or with cell constituents such as plasma membrane glycoproteins. Such enhanced interaction between the cationic transfection lipid and the biological by active molecules and/or cell membrane constituents may play a key role in successfully transporting the therapeutic molecules into the cells.
The cationic amphiphiles of the present invention have certain common structural and functional groups. As such, the said cationic amphiphilic compounds may be represented by the following generic formula: 
wherein
n is an integer between 1 and 3,
R1 may be H, saturated or unsaturated aliphatic group (C8-C20) or long chain saturated or unsaturated alkyl group (C7-C19).
R3 may be hydroxyalkyl group of 1-3 carbon atoms,
R2 may be a long chain saturated alkyl group (C7-C19) or [(CH2)nxe2x80x94Zxe2x80x94R2]
Z may be methylene group (xe2x80x94CH2), or an esteric group (xe2x80x94Oxe2x80x94COxe2x80x94)
Various novel amphiphilic compounds having the above basic structural formula are described in co-pending U.S. patent application Ser. No. 09/275,816 (Indian Application No.3324/DEL/98, 3325/DL/98 and 3327/DEL/98 all dated 9th Nov, 1998). Further, in the said amphihpiles the preference of C8-C20 is specifically low because below the chain length of C8 the amphiphiles loose the aggregation property and the specific reason for not using carbon chains longer than C20 is because they are not compatible with biological membranes in terms of their lengths. Further, the additional linking groups that may be practised within the scope of this invention include xe2x80x94Oxe2x80x94(ether) CONH-(amide) group etc. Among the three different linking groups described in the invention herein below, the esteric group is most preferred
Further, the products obtained during the synthesis of the said amphiphilic compounds are hydrolysis products. The molecules were so constructed to aid in their breakdown within a cell subsequent to performing the task of transfection. The structure of the amphiphiles may also be altered by a combination of alkyl and amine moieties, which structures would fall within the teachings and scope of the invention. Such modifications are apparent to those skilled in the art.
Accordingly, the invention provides cationic amphiphiles represented by the following structural formula (I): 
wherein:
n is an integer between 1 and 3;
R1 represents either H or a saturated aliphatic group;
R2 independently represents a long chain saturated alkyl group (from C7 to C19);
R3 is a small hydroxyalkyl group containing 1-3 carbon atoms;
X is either a halogen atom or a tosylate group.
Z represents a methylene (xe2x80x94CH2xe2x80x94) group;
The process for the synthesis of said cationic amphiphilic compounds having the above structural formula (I) comprises reacting the secondary amine containing the N-hydroxyalkyl group such as diethanolamine with a saturated alkyl halides or saturated alkyl tosylates in a polar solvent in the presence of a weak tertiary base.
In an embodiment of the invention, the N-hydroxyalkyl group may be diethanolamine.
In another embodiment, the saturated alkyl bromides employed in the process may be selected from the group consisting of n-hexadecyl bromide.
In yet another embodiment, the polar solvents in which the reaction may be carried out may be selected from the group comprising alcohols, diemethyl formamide, dimethylacetamide, acetonitrile, methylisobutyl ketone.
In still another embodiment of the invention, the base may be selected from sodium or potassium carbonate.
In yet another embodiment, the reaction for the synthesis of said amphiphilic lipids may be carried out at a temperature in the range of 0xc2x0-250xc2x0 C., preferably at the reflux temperature (boiling point of the solvent selected).
According to an aspect of the invention, a pair of particularly effective representative cationic amphiphiles that may be synthesised by the said reaction, would include N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB), amphiphile No 1 and N-n-hexadecyl-N,N-dydroxyethylammonium bromide (HDEAB), amphiphile No. 2 represented as under: 
The process for the synthesis of said cationic lipids is schematically represented hereunder. 
The invention further provides cationic amphiphilic compounds represented by the structural formula (II): 
wherein:
n is an integer between 1 and 3;
R1 independently, represents either a saturated aliphatic group or an unsaturated aliphatic group (from C8 to C20);
Z represents a methylene (xe2x80x94H20xe2x80x94) group;
R2, independently, represents a long-chain saturated alkyl group (form C7 to C19);
R3 is a small alkyl group (from C1 to C3);
X is either a halogen atom or a tosylate group.
The process for the synthesis of said cationic amphiphilic compounds having the above structural formula,comprises:
(a) coupling the aliphatic unsaturated aldehyde with the alkyl amine followed by the reduction of the resulting imine to obtain the corresponding secondary amine;
(b) reacting the secondary amine obtained in step (a) with the hydroxyl-protected hydroxyalkyl halide followed by removal of the hydroxyl protecting group to obtain the corresponding N-hydroxyalyl group containing tertiary amine and
(c) quaternizing the resulting N-hydroxyalkyl group containing tertiary amine obtained in step (b) with an alkyl halide or alkyl tosylates in a mixed polar solvent.
In an embodiment of the invention, the saturated or unsaturated aldehyde used may be oleyl aldehyde.
In another embodiment, the alkyl amine used may be n-octadecyl amine.
In yet another embodiment, the tertiary amine may be quartemized with alkyl iodide selected from methyl iodide.
In yet another embodiment, the mixed polar solvent may comprise a mixture of methanol and chloroform.
In yet another embodiment, the resultant tertiary amine of step (b) may be N-hydroxyethyl-N-oleyl-N-n-octadecyl-amine
In an embodiment, the reaction of amnie with the aldehyde in step (a) is generally performed in a dry chlorinated solvent such as dichloromethane with temperature in the range of xe2x88x9278xc2x0 C. to 10xc2x0 C. and the reduction of the resulting intermediate imine is carried out with sodium borohydride in a mixed polar solvent such as methanol and dichloromethane with temperature in the range of xe2x88x925xc2x0 C. to 40xc2x0 C. The step (b) is generally carried out in a polar solvent such as ethyl acetate, N,N-dimethyl formamide, acetonitrile and the like in the presence of a weak base such as sodium or potassium carbonate. The solvent selected in removing the hydroxyl protecting group in step (b) is generally polar aprotic in nature such as tetrahydrofliran, dinethyl formamide and the like. The final quartemization in step (c) is carried out using the appropriate alkyl polar solvent such as methanol and chloroform at a temperature in the range xe2x88x925xc2x0 C. to 40xc2x0 C.
According to this aspect of the invention, a particularly effective representative pair of cationic transfection lipids prepared by the above process would include, for example, N-methyl-N-n-octadecyl-N-oleyl-N-hydroxyethyl-ammonium chloride (MOOHAC), amphiphile No 3 and N,N-di-n-octadecyl-N-methyl-N-dihydroxyethylammonium chloride (DOMHAC), amphiphile No. 4, whose structural formulae are represented hereunder: 
A schematic representation of the process described above for the synthesis of said cationic amphiphiles is provided hereunder for reference: 
The invention further provides a process for the preparation of cationic amphiphiles represented by the structural formula (III) given hereunder: 
wherein:
n is an integer between 1 and 3, Z an ester group (xe2x80x94Oxe2x80x94COxe2x80x94) and R1 and R2 independently, represent a long-chain saturated or unsaturated alkyl group (from C7 to C19), R3 is a small alkyl group (C1 to C3) and X is either a halogen atom or a tosylate group.
The process for the synthesis of said cationic amphiphilic compounds, comprises:
(a) reacting an acid chloride with a tertiary amine containing the N,N-dihydroxyalkyl group to obtain the hydrochloride salt of the corresponding di-O-acylated product,
(b) neutralizing the resulting hydrochloride salt obtained in step (a) with alkali and
(c) quatemizing the resulting tertiary amine obtained in step (b) with the appropriate hydroxy-alkyl halide.
In an embodiment of the invention, the acid chloride used in step (a) may be n-hexadecanoyl chloride.
In another embodiment, the tertiary amine in step (a) may be N-methyldiethanolamine.
In yet another embodiment, the reaction in step (a) may be performed at a temperature in the range of 10xc2x0-50xc2x0 C., in a polar aprotic solvent selected from the group comprising N,N-dimethylformamide, acetonitrile and the like.
In yet another embodiment, the neutralization in step (b) may be carried out in a biphasic solvent such as mixture of dichloromethane and water or ethyl acetate and water using strong alkali such as NaOH or KOH.
In yet another embodiment, the quaternisation in step (c) is carried out at a temperature in the range of 40xc2x0 C. to 100xc2x0 C. depending on the nature of alkyl halides used.
In another embodiment, the hydroxyalkyl halide used in step (a) is 2-bromoethanol.
According to this aspect of the invention, a particularly effective cationic transfection lipid that may be prepared by the above process includes-N,N,-di[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N-methylammonium bromide (DOHEMAB), amphiphile 5 having the structural formula as hereunder: 
The said process for the synthesis of cationic lipids is schematically represented hereunder: 